Duchenne muscular dystrophy (DMD) is a progressive disease characterized by the lack of dystrophin under the sarcolemmal membrane6,19,28,37. One possible way to introduce dystrophin in the muscle fibers of the patients to limit the degeneration is to transplant myoblasts obtained from normal subjects30,34,35. Several groups have tried myoblast transplantations to DMD patients but poor graft success was observed17,22,24,38. Even in experimental myoblast transplantation using mdx mice, an animal model of DMD10,25,29, large amount of dystrophin-positive fibers were observed only when nude mdx mice were previously irradiated to prevent regeneration of the muscle fibers by host myoblasts32,43. High percentage of dystrophin-positive fibers was also observed in mdx mice immunosuppressed with FK 506 and in SCID mice, in both cases muscles were previously damaged by notexin injection and irradiated23,27. These results indicate that to obtain successful myoblast transplantation, it is necessary to have not only an immunodeficient mouse or a mouse adequately immunosuppressed but also a host muscle which has been adequately preconditioned. It is, however, impossible in clinical studies to use damaging treatments such as marcaine, notexin and irradiation. If good myoblast transplantation results can be obtained without using such techniques, this would be very helpful for myoblast transplantation in humans.
Recently there has been an increasing interest on the effects of basic fibroblast growth factor (bFGF) and other growth factors on myoblast cultures and myoblast cell lines1,4,5. Basic FGF has been reported to both stimulate proliferation and inhibit differentiation of skeletal myoblasts in vitro15,16. Other growth or trophic factors like insulin growth factor I, transferrin, platelet-derived growth factor, epidermal growth factor, adrenocorticotrophin and macrophage colony-stimulating factor as well as C kinase proteins activators or agonists by which the effect of bFGF is mediated20 may also have similar or even better effects than bFGF on the success of myoblast transplantation7. The use of these stimulating properties to enhance the success of transplantation by in vitro preconditioning of donor's cells and to replace at least partially the use of previously known methods of in vivo preconditioning of recipients' cells has never been suggested before.
Furthermore, it has been recently published by Overall and Sodek (1996) that concanavalin A increased the secretion of metalloproteases by fibroblasts. Since these enzymes are believed to be present in primary myoblasts cultures, and since they may be responsible for the degradation of the extracellular matrix, it would be desirable to precondition the myoblasts in the presence of both a growth factor and an inducer of the production of metalloproteases, to increase the distance of migration of the transplanted myoblasts and to increase the number of fused myoblasts expressing muscle functional proteins. An attractive alternative would be to use donor myoblasts wherein a gene expressing a metalloprotease is inserted.
Metalloproteases are enzymes necessary for tumor invasion, for cell migration45, and for restructuration of extracellular matrix during normal tissue remodelization46. Matrilysine and gelatinase A are metalloproteases involved in tissue invasion of a plurality of cancer types47. The presence of gelatinase A in its active form has been correlated with the generation of new muscle fibers, during muscle degeneration-regeneration process48. It has been shown that the activity of gelatinase A can induce cell migration by cleaving laminin-5, an extracellular matrix component, thereby exposing a pro-migratory kryptic site49.
From the foregoing, it is really apparent that a compound capable of stimulating the expression of a metalloproteases involved in an extra-cellular restructuration, such as phorbol ester or concanavalin A, would be useful to increase the success of transplantation of myoblasts. Since metalloproteases appear to be secreted in the culture medium, it would also be useful to test if metalloproteases such as matrilysine, gelatinase A, or other metalloproteases of the same class, could be injected directly with myoblasts in recipient muscle for the same purpose.